It is clear that HIV-1 can establish an independently replicating population in the CNS, and that this can also lead to the evolution of macrophage-tropic HIV-1, defined as the ability to enter cells with low levels of CD4. However, the distributin of infected cells in the brain and the occurrence of macrophage-tropic virus outside of the CNS are both controversial and both of these points are important considerations in defining strategies for the eradication of HIV-1. Our understanding of viral populations has been significantly improved with the recent widespread acknowledgement that PCR-mediated recombination can scramble phylogenetic information, and that the use of template end-point dilution in PCR (single genome amplification) is essential to define viral populations accurately. Similarly, improvements in programs modeling evolutionary processes make the assessment of phylogenetic relationships more robust, for example incorporating the Bayesian analysis program BEAST. Finally, quantitative descriptions of CD4 dependence for cell entry provide a much more robust definition of macrophage tropism than using highly variable monocyte-derived macrophage preparations to try to phenotype viruses that have evolved to use low levels of CD4 to enter cells. We are applying all three of these important advances to understand the evolution of macrophage-tropic viruses and their role in pathogenesis and to understand their potential to create a long-lived reservoir. We have recently brought these tools together to study viral compartmentalization, including compartmentalization in the CNS by examining virus in the CSF. We have now extended these studies to include samples from the National NeuroAIDS Tissue Consortium (NNTC). We propose to exploit the availability of these samples, taken both prior to and at autopsy, to map the sites of viral replication in the brain in subjects how are viremic and those on therapy. We will also define the role of macrophage-tropic virus in populating centers of replication in the brain, and the ability of this type of evolutionary variant to become established outside of the CNS. Finally, we will examine the phenotype of the early rebound virus that appears after therapy discontinuation, as this represents the first virus that eradication strategies must confront.